Establishment And Primary Application Of Gene Chip For Detecting Chikungunya Virus And Sindbis Virus

Chikungunya (CHIK) fever and Sindbis (SIND) fever are both natural epidemic zoonosis, which cause diseases spreading mainly through sensitive vertebrates which are bite by blood-sucking arthropod. For Chikungunya fever and Sindbis fever are widely distributed in the world, invasion and spread rapidly, resulting in international trade barriers and huge economic losses, seriously threaten the public health and safety of the world, even lead to direct infection and death of the human. At present, no cure drugs worked for both diseases, early and effective diagnosis is the important measures for prevention and mitigation of the spread of the both diseases.Chikungunya virus (CHIKV) and Sindbis virus (SINDV) are commonly detected with methods of virus isolation, serological tests, antigen detection and multiplex PCR detection. However, CHIKV and SINDV are both belong to the insect-borne virus, share the extremely similar biological structure, and cause almost no difference in Clinical symptoms, no precise identification was accomplished with detection method above. Therefore, an efficient, high-throughput and rapid detection method to distinguish these two viruses is necessary to be found out. Gene chip technology is a highly chiasmal leading edge technology developed in recent years mixed with biology, physics, chemistry, computer science and microelectronics, not only has the advantages of rapid, sensitive, specific, high-throughput and automation, but also improve the reliability in results determine for data, and results are determined all by computer software, is forecasted a very broad application prospects in the rapid differential diagnosis and detection. Gene chip technology provides a possible way for CHIKV and SINDV detection at the same time. According to the differences between CHIKV and SINDV of gene sequences, This study used gene chip technology, multiplex PCR, and nucleic acid labeling technique simultaneously, building a detection of gene chips of CHIKV and SINDV for CHIKV and SINDV identification.First, the genome sequences of CHIKV and SINDV from different areas, different countries were downloaded from NCBI database, then were analysis systemically in use of molecular biology software, to find consistent sequences between CHIKV and SINDV genome, respective reference E gene of CHIKV, SINDV were synthesized with CHIKV Asian strains DQ443544and SINDV South Africa-strain ACU38305, the result showed E gene nucleotide Sequence was homology between CHIKV and SINDV, which provide test material for gene chip detection.In addition, based on the results of sequence analysis,2multiplex PCR primers for CHIKV and SINDV detection,5specific oligonucleotide probes,1positive coordinate probe,1probe to monitoring print and post processing were designed with the application of bioinformatics software. Under the same condition, microarray hybridized JEV Ⅰ, JEV Ⅲ, PRRSV and AIV with chip after processing, and then scanned the chip to detect the susceptiveness. Microarray hybridized the pMD-CHIK, the pMD-of SIND plasmid DNA after multiproportion dilution and then scanned the chip to detect the susceptiveness. The results showed that the chip prepared in this experiment-specific negative control of JEV Ⅰ of JEV Ⅲ, PRRSV and AIV found no signal detected as a negative control, and had good repetitiveness. The sensitivity of the experimental results showed that the determination of sample size1.0ng/ml was the lowest test line. Experimental evaluations proved this diagnostic chip in good reproducibility, and could be saved at least two months at room temperature conditions without affecting the detection results.